Laing early-onset distal myopathy with subsarcolemmal hyaline bodies caused by a novel variant in the MYH7 gene.

Myopathies caused by MYH7 gene mutations are clinically and pathologically heterogeneous and, until recently, difficult to diagnose. The availability of NGS panels for hereditary neuromuscular diseases changed our insight regarding their frequency and allowed a better perception of the different phenotypes and morphological abnormalities associated. We present a male Portuguese patient with the classical phenotype of Laing early-onset distal myopathy (MPD1) beginning at 6 years of age, very slowly progressive, and with a mild to moderate impact on daily life by the age of 56. Muscle biopsy showed a myopathic pattern with hyaline bodies and cores. The NGS panel for structural myopathies identified a novel missense heterozygous variant, c.T4652C (p.Leu1551Pro), in the exon 34 of the MYH7 gene.

PattRec: An easy-to-use CNV detection tool optimized for targeted NGS assays with diagnostic purposes.

Genetic laboratories use custom-commercial targeted next-generation sequencing (tg-NGS) assays to identify disease-causing variants. Although the high coverage achieved with these tests allows for the detection of copy number variants (CNVs), which account for an important proportion of the genetic burden in human diseases, an easy-to-use tool for automatic CNV detection is still lacking. This article presents a new CNV detection tool optimized for tg-NGS data: PattRec. PattRec was evaluated using a wide range of data, and its performance compared with those of other CNV detection tools. The software includes features for selecting optimal controls, discarding polymorphic CNVs prior to analysis, and filtering out deletions based on SNV zygosity, and automatically creates an in-house CNV database. There is no need for high level bioinformatic expertise and users can choose color-coded xlsx output that helps to prioritize potentially pathogenic CNVs. PattRec is presented as a Java based GUI, freely available online: https://github.com/irotero/PattRec.

Rare Variants in 48 Genes Account for 42% of Cases of Epilepsy With or Without Neurodevelopmental Delay in 246 Pediatric Patients.

In order to characterize the genetic architecture of epilepsy in a pediatric population from the Iberian Peninsula (including the Canary Islands), we conducted targeted exome sequencing of 246 patients with infantile-onset seizures with or without neurodevelopmental delay. We detected 107 variants in 48 different genes, which were implicated in neuronal excitability, neurodevelopment, synaptic transmission, and metabolic pathways. In 104 cases (42%) we detected variant(s) that we classified as pathogenic or likely pathogenic. Of the 48 mutated genes, 32 were dominant, 8 recessive and 8 X-linked. Of the patients for whom family studies could be performed and in whom pathogenic variants were identified in dominant or X-linked genes, 82% carried de novo mutations. The involvement of small copy number variations (CNVs) is 9%. The use of progressively updated custom panels with high mean vertical coverage enabled establishment of a definitive diagnosis in a large proportion of cases (42%) and detection of CNVs (even duplications) with high fidelity. In 10.5% of patients we detected associations that are pending confirmation via functional and/or familial studies. Our findings had important consequences for the clinical management of the probands, since a large proportion of the cohort had been clinically misdiagnosed, and their families were subsequently able to avail of genetic counseling. In some cases, a more appropriate treatment was selected for the patient in question, or an inappropriate treatment discontinued. Our findings suggest the existence of modifier genes that may explain the incomplete penetrance of some epilepsy-related genes. We discuss possible reasons for non-diagnosis and future research directions. Further studies will be required to uncover the roles of structural variants, epimutations, and oligogenic inheritance in epilepsy, thereby providing a more complete molecular picture of this disease. In summary, given the broad phenotypic spectrum of most epilepsy-related genes, efficient genomic tools like the targeted exome sequencing panel described here are essential for early diagnosis and treatment, and should be implemented as first-tier diagnostic tools for children with epilepsy without a clear etiologic basis.

A de novo heterozygous missense BSCL2 variant in 2 siblings with intractable developmental and epileptic encephalopathy.

PURPOSE: We present the case of 2 siblings with profound refractory epilepsy and neurological regression that began at the ages of 3 and 6 months. Diagnosis remained elusive despite extensive metabolic and genetic workups, including use of a targeted next-generation sequencing panel for epilepsy genes. METHODS: Whole-exome sequencing was performed for the 2 siblings and their unaffected parents, in addition to fibroblast cell culture, RNA extraction and reverse-transcription, and cDNA PCR. Brain tissue from one of the siblings was collected post-mortem for neuropathological examination, including histology and immunohistochemistry. RESULTS: Ade novo nucleotide change (c.566 T > A; p.(Met189Lys)) in exon 4 of the BSCL2 gene was detected in the 2 siblings, and confirmed by Sanger sequencing. This variant was absent in the parents and in a third, unaffected sibling. CONCLUSION: Given thede novo nature of the variant, its absence from public and in-house databases, our in silico pathogenicity predictions, and co-segregation of the variant with the disease phenotype, we believe that this novel variant is associated with the epileptic encephalopathy phenotype of the 2 siblings. Our findings provide the first evidence of an association between a heterozygous BSCL2 variant and developmental and early infantile epileptic encephalopathy. Further functional studies will be needed to elucidate the pathophysiological mechanisms underlying this new BSCL2-associated phenotype.

Free-access copy-number variant detection tools for targeted next-generation sequencing data.

Copy number variants (CNVs) are intermediate-scale structural variants containing copy number changes involving DNA fragments of between 1 kb and 5 Mb. Although known to account for a significant proportion of the genetic burden in human disease, the role of CNVs (especially small CNVs) is often underestimated, as they are undetectable by traditional Sanger sequencing. Since the development of next-generation sequencing (NGS) technologies, several research groups have compared depth of coverage (DoC) patterns between samples, an approach that may facilitate effective CNV detection. Most CNV detection tools based on DoC comparisons are designed to work with whole-genome sequencing (WGS) or whole-exome sequencing (WES) data. However, few methods developed to date are designed for custom/commercial targeted NGS (tg-NGS) panels, the assays most commonly used for diagnostic purposes. Moreover, the development and evaluation of these tools is hindered by (i) the scarcity of thoroughly annotated data containing CNVs and (ii) a dearth of simulation tools for WES and tg-NGS that mimic the errors and biases encountered in these data. Here, we review DoC-based CNV detection methods described in the current literature, assess their performance with simulated tg-NGS data, and discuss their strengths and weaknesses when integrated into the daily laboratory workflow. Our findings suggest that the best methods for CNV detection in tg-NGS panels are DECoN, ExomeDepth, and ExomeCNV. Regardless of the method used, there is a need to make these programs more user-friendly to enable their use by diagnostic laboratory staff who lack bioinformatics training.

Mutación puntual de novo en el gen KCND3 en un paciente con ataxia crónica de inicio precoz / De novo sporadic mutation in the KCND3 gene in a patient with early onset chronic ataxia

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Bethlem myopathy: a series of 16 patients and description of seven new associated mutations.

BACKGROUND: Bethlem myopathy represents the milder phenotype of collagen type VI-related myopathies. However, clinical manifestations are highly variable among patients and no phenotype-genotype correlation has been described. We aim to analyse the clinical, pathological and genetic features of a series of patients with Bethlem myopathy, and we describe seven new mutations. METHODS: A series of 16 patients with the diagnosis of Bethlem myopathy were analyzed retrospectively from their medical records for clinical, creatine kinase (CK), muscle biopsy, and muscle magnetic resonance (MRI) data. Genetic testing was performed through next-generation sequencing of custom amplicon-based targeted genes panel of myopathies. Mutations were confirmed by Sanger sequencing. RESULTS: The most frequent phenotype consisted of proximal limb weakness associated with interphalangeal and wrists contractures. However, cases with isolated contractures or isolated myopathy were found. CK levels did not correlate with severity of the disease. The most frequent mutation was the COL6A3 variant c.7447A>G, p.Lys2486Glu, with either an homozygous or compound heterozygous presentation. Five new mutations were found in COL6A1 gene and other two in COL6A3 gene, all of them with a dominant heritability pattern. From these, a new COL6A1 mutation (c.1657G>A, p.Glu553Arg) was related to an oligosymptomatic phenotype with predominating contractures in the absence of weakness and a normal muscle MRI. Finally, the most common COL6A1 mutation reported to date that leads to an Ullrich phenotype (c. 868G>A, p.Gly290Arg), has been found here as Bethlem presentation. CONCLUSIONS: Manifestations of Bethlem myopathy are quite variable, so either contractures or weakness may be lacking, and no phenotype-genotype associations can be brought.

Molecular Characterization of New FBXL4 Mutations in Patients With mtDNA Depletion Syndrome.

Encephalomyopathic mitochondrial DNA (mtDNA) depletion syndrome 13 (MTDPS13) is a rare genetic disorder caused by defects in F-box leucine-rich repeat protein 4 (FBXL4). Although FBXL4 is essential for the bioenergetic homeostasis of the cell, the precise role of the protein remains unknown. In this study, we report two cases of unrelated patients presenting in the neonatal period with hyperlactacidemia and generalized hypotonia. Severe mtDNA depletion was detected in muscle biopsy in both patients. Genetic analysis showed one patient as having in compound heterozygosis a splice site variant c.858+5G>C and a missense variant c.1510T>C (p.Cys504Arg) in FBXL4. The second patient harbored a frameshift novel variant c.851delC (p.Pro284LeufsTer7) in homozygosis. To validate the pathogenicity of these variants, molecular and biochemical analyses were performed using skin-derived fibroblasts. We observed that the mtDNA depletion was less severe in fibroblasts than in muscle. Interestingly, the cells harboring a nonsense variant in homozygosis showed normal mtDNA copy number. Both patient fibroblasts, however, demonstrated reduced mitochondrial transcript quantity leading to diminished steady state levels of respiratory complex subunits, decreased respiratory complex IV (CIV) activity, and finally, low mitochondrial ATP levels. Both patients also revealed citrate synthase deficiency. Genetic complementation assays established that the deficient phenotype was rescued by the canonical version of FBXL4, confirming the pathological nature of the variants. Further analysis of fibroblasts allowed to establish that increased mitochondrial mass, mitochondrial fragmentation, and augmented autophagy are associated with FBXL4 deficiency in cells, but are probably secondary to a primary metabolic defect affecting oxidative phosphorylation.

Congenital hyperinsulinism in two siblings with ABCC8 mutation: same genotype, different phenotypes.

Congenital hyperinsulinism (CHI) is a heterogenous disease caused by insulin secretion regulatory defects, being ABCC8/KCNJ11 the most commonly affected genes. Therapeutic options include diazoxide, somatostatin analogues and surgery, which is curative in focal CHI. We report the case of two siblings (born two years apart) that presented themselves with hypoketotic hyperinsulinemic persistent hypoglycemias during neonatal period. The diagnosis of diffuse CHI due to an ABCC8 compound mutation (c.3576delG and c.742C>T) was concluded. They did not benefit from diazoxide therapy (or pancreatectomy performed in patient number 1) yet responded to somatostatin analogues. Patient number 1 developed various neurological deficits (including epilepsy), however patient number 2 experienced an entirely normal neurodevelopment. We believe this case shows how previous knowledge of the firstborn sibling's disease contributed to a better and timelier medical care in patient number 2, which could potentially explain her better neurological outcome despite their same genotype.

Congenital hyperinsulinism in two siblings with ABCC8 mutation: same genotype, different phenotypes

SUMMARY Congenital hyperinsulinism (CHI) is a heterogenous disease caused by insulin secretion regulatory defects, being ABCC8/KCNJ11 the most commonly affected genes. Therapeutic options include diazoxide, somatostatin analogues and surgery, which is curative in focal CHI. We report the case of two siblings (born two years apart) that presented themselves with hypoketotic hyperinsulinemic persistent hypoglycemias during neonatal period. The diagnosis of diffuse CHI due to an ABCC8 compound mutation (c.3576delG and c.742C>T) was concluded. They did not benefit from diazoxide therapy (or pancreatectomy performed in patient number 1) yet responded to somatostatin analogues. Patient number 1 developed various neurological deficits (including epilepsy), however patient number 2 experienced an entirely normal neurodevelopment. We believe this case shows how previous knowledge of the firstborn sibling's disease contributed to a better and timelier medical care in patient number 2, which could potentially explain her better neurological outcome despite their same genotype.

SIGMAR1 gene mutation causing Distal Hereditary Motor Neuropathy in a Portuguese family.

SIGMAR1 gene encodes a non-opioid endoplasmic reticulum (ER) protein which is involved in a large diversity of cell functions and is expressed ubiquitously in both central and peripheral nervous systems. Alterations of its normal function may contribute to two different phenotypes: juvenile amyotrophic lateral sclerosis (ALS 16) and distal hereditary motor neuropathies (dHMN). We present the case of a female patient, of 37-years-old, with distal muscle weakness and atrophy beginning in childhood and slowly progressive in the first two decades of life. Neurological examination revealed a symmetrical severe muscle wasting and weakness in distal lower and upper limbs, with claw hands, footdrop with equinovarus deformity and hammer toes, generalized areflexia and normal sensory examination. The electrodiagnostic study revealed a pure chronic motor peripheral nerve involvement without signs of demyelination. The molecular study found the deletion c.561_576del on exon 4 and a deletion of all exon 4, in the SIGMAR1 gene.

Novel mosaic mutation in the dystrophin gene causing distal asymmetric muscle weakness of the upper limbs and dilated cardiomyopathy.

A group of heterogeneous muscle diseases are caused by dystrophin gene (DMD) mutations. We hereby present a male patient with a diagnosis of symptomatic dilated cardiomyopathy at 44 years-old who developed, soon after, weakness of distal right upper limb. At the age of 58, neurological examination revealed severe atrophy of right thenar muscles, flexion contractures on the right elbow, wrist and fingers, bilateral calf hypertrophy, myotatic areflexia in the upper limbs and hyporeflexia in the lower limbs. Manual muscle examination showed distal weakness of right upper limb muscles, severe on abductor pollicis brevis and extensor pollicis longus, and milder on interossei, finger extensors and brachioradialis muscles. Further testing revealed CK of 1500 U/L, a myopathic pattern on electromyography, and myopathic changes on right deltoid muscle biopsy, with immunohistochemistry showing focal sub-expression of dystrophin. Cardiac workup revealed a severe reduction in left ventricular ejection fraction, with a left ventricle of increased dimensions and global hypo-contractibility. A next-generation sequencing based panel for muscular diseases was performed and a nonsense mutation (c.C7525T) was identified in exon 51 of DMD gene, present in 70% of the gene readings (consistent with mosaicism).

Carbohydrate status in patients with phenylketonuria.

BACKGROUND: In patients with phenylketonuria (PKU), a low-phenylalanine (Phe) diet supplemented with low-protein foods and a Phe-free amino acid mixture favors a dietary intake rich in carbohydrates, but little is known about how these molecules are metabolized in this setting. The objective of the present study was to analyze carbohydrate metabolism in patients with hyperphenylalaninemia. METHODS: We conducted a multicenter cross-sectional study to investigate biochemical markers of basal and postprandial carbohydrate metabolism in PKU patients according to age, Phe tolerance, waist circumference and body mass index (BMI), diet, tetrahydrobiopterin (BH4) supplementation, and adherence to treatment. Basal biomarkers and anthropometric parameters were also evaluated in patients with mild hyperphenylalaninemia (MHPA) and in healthy controls. RESULTS: A total of 83 patients aged 4-52 years were studied; 68.7% had PKU and 31.3% had MHPA. 68 healthy controls of similar sex and age were also evaluated Metabolic control was adequate in 71.9% of PKU patients. Fasting glucose levels (mean 80.77 ± 8.06 mg/dL) were high in just one patient, but fasting insulin levels, with a mean of 12.74 ± 8.4 mIU/L, were altered in 15 PKU patients (26.3%) and markedly higher than in patients with MPHA (p = 0.035). Fasting insulin levels and Homeostasis Model Assessment Insulin Resistance (HOMA-IR) were significantly higher than in healthy controls and correlated with body mass index, waist circumference, age, and also showed statistically significant differences according to diagnosis and Phe tolerance (p < 0.05). Patients under BH4 therapy had lower insulin levels and HOMA-IR. A higher mean carbohydrate intake from AA mixtures was observed in classic PKU patients. The caloric intake in the form of carbohydrates was also higher in PKU than MHPA patients (p = 0.038) and it was correlated with basal insulin (rho = 0.468, p = 0.006), HOMA-IR (rho = 0.423, p = 0.02), BMI (rho 0.533, p = 0.002), and waist circumference (rho 0.584, p = 0.0007). CONCLUSIONS: This study shows that PKU patients are at risk of carbohydrate intolerance and insulin resistance, more evident in adults and overweight patients, probably related to their higher caloric intake in form carbohydrate content. A higher dependency of AA mixtures was demonstrated in PKU patients.

Prioritization of Variants Detected by Next Generation Sequencing According to the Mutation Tolerance and Mutational Architecture of the Corresponding Genes.

The biggest challenge geneticists face when applying next-generation sequencing technology to the diagnosis of rare diseases is determining which rare variants, from the dozens or hundreds detected, are potentially implicated in the patient's phenotype. Thus, variant prioritization is an essential step in the process of rare disease diagnosis. In addition to conducting the usual in-silico analyses to predict variant pathogenicity (based on nucleotide/amino-acid conservation and the differences between the physicochemical features of the amino-acid change), three important concepts should be borne in mind. The first is the "mutation tolerance" of the genes in which variants are located. This describes the susceptibility of a given gene to any functional mutation and depends on the strength of purifying selection acting against it. The second is the "mutational architecture" of each gene. This describes the type and location of mutations previously identified in the gene, and their association with different phenotypes or degrees of severity. The third is the mode of inheritance (inherited vs. de novo) of the variants detected. Here, we discuss the importance of each of these concepts for variant prioritization in the diagnosis of rare diseases. Using real data, we show how genes, rather than variants, can be prioritized by calculating a gene-specific mutation tolerance score. We also illustrate the influence of mutational architecture on variant prioritization using five paradigmatic examples. Finally, we discuss the importance of familial variant analysis as final step in variant prioritization.

A novel missense mutation in GRIN2A causes a nonepileptic neurodevelopmental disorder.

BACKGROUND: Mutations in the GRIN2A gene, which encodes the GluN2A (glutamate [NMDA] receptor subunit epsilon-1) subunit of the N-methyl-d-aspartate receptor, have been identified in patients with epilepsy-aphasia spectrum disorders, idiopathic focal epilepsies with centrotemporal spikes, and epileptic encephalopathies with severe developmental delay. However, thus far, mutations in this gene have not been associated with a nonepileptic neurodevelopmental disorder with dystonia. OBJECTIVES: The objective of this study was to identify the disease-causing gene in 2 siblings with neurodevelopmental and movement disorders with no epileptiform abnormalities. METHODS: The study method was targeted next-generation sequencing panel for neuropediatric disorders and subsequent electrophysiological studies. RESULTS: The 2 siblings carry a novel missense mutation in the GRIN2A gene (p.Ala643Asp) that was not detected in genomic DNA isolated from blood cells of their parents, suggesting that the mutation is the consequence of germinal mosaicism in 1 progenitor. In functional studies, the GluN2A-A643D mutation increased the potency of the agonists L-glutamate and glycine and decreased the potency of endogenous negative modulators, including protons, magnesium and zinc but reduced agonist-evoked peak current response in mammalian cells, suggesting that this mutation has a mixed effect on N-methyl-d-aspartate receptor function. CONCLUSION: De novo GRIN2A mutations can give rise to a neurodevelopmental and movement disorder without epilepsy. © 2018 International Parkinson and Movement Disorder Society.

NGS Technologies as a Turning Point in Rare Disease Research , Diagnosis and Treatment.

Approximately 25-50 million Americans, 30 million Europeans, and 8% of the Australian population have a rare disease. Rare diseases are thus a common problem for clinicians and account for enormous healthcare costs worldwide due to the difficulty of establishing a specific diagnosis. In this article, we review the milestones achieved in our understanding of rare diseases since the emergence of next-generation sequencing (NGS) technologies and analyze how these advances have influenced research and diagnosis. The first half of this review describes how NGS has changed diagnostic workflows and provided an unprecedented, simple way of discovering novel disease-associated genes. We focus particularly on metabolic and neurodevelopmental disorders. NGS has enabled cheap and rapid genetic diagnosis, highlighted the relevance of mosaic and de novo mutations, brought to light the wide phenotypic spectrum of most genes, detected digenic inheritance or the presence of more than one rare disease in the same patient, and paved the way for promising new therapies. In the second part of the review, we look at the limitations and challenges of NGS, including determination of variant causality, the loss of variants in coding and non-coding regions, and the detection of somatic mosaicism variants and epigenetic mutations, and discuss how these can be overcome in the near future.

Homozygous truncating mutation in prenatally expressed skeletal isoform of TTN gene results in arthrogryposis multiplex congenita and myopathy without cardiac involvement.

We report the case of a newborn with arthrogryposis multiplex congenita and severe axial hypotonia without cardiac involvement in which, using a customized targeted next-generation sequencing assay for 64 myopathy-associated genes, we detected a novel homozygous truncating mutation, c.38661_38665del, in exon 197 of the TTN gene that is expressed only in the fetal skeletal isoform. Its pathogenicity is supported by evidence of maternal isodisomy for chromosome 2. Muscle pathology showed fibers with core-like areas devoid of oxidative staining and cytoplasmic bodies. Electron microscopy showed the replacement of the sarcomeric structure with filamentous material. Identification of this mutation expands the phenotypic spectrum of the TTN gene and shows for the first time that a mutation not found in adult TTN isoforms is involved in the development of a neuromuscular disorder. TTN mutations should be considered in all severe congenital myopathies with arthrogryposis without cardiac involvement.

Clinical and genetic features of 13 Spanish patients with KCNQ2 mutations.

The KCNQ2 gene codifies a subunit of the voltage-gated potassium M channel underlying the neuronal M-current. Classically, mutations in this gene have been associated with benign familial neonatal seizures, however, in recent years KCNQ2 mutations have been reported associated to early-onset epileptic encephalopathy. In this work, detailed familiar, clinical and genetic data were collected for 13 KCNQ2-positive patients revealed among a cohort of 80 epileptic pediatric probands from Spain who were analyzed through a targeted next-generation sequencing assay for 155 epilepsy-associated genes. This work shows for the first time the association between KCNQ2 mutations and startle attacks in 38% of patients, which opens the possibility to define electroclinical phenotypes associated to KCNQ2 mutations. It also demonstrates that KCNQ2 mutations contribute to an important percentage of Spanish patients with epilepsy. The study confirm the high genetic heterogeneity of this gene with 13 different mutations found, 10 of them novel and the better outcome of patients treated with sodium channel blockers.

Genotype and phenotype characterization in a Spanish cohort with isovaleric acidemia.

Isovaleric acidemia (IVA) is a rare disorder of leucine metabolism. We carried out a multicenter study of IVA patients diagnosed by newborn screening (NBS) or symptoms clinics over a period of 28 years in Spain. Evaluated at diagnosis, data included age, detection method, levels of C5 and IVG, enzymatic studies, clinical presentation parameters and genotype in 16 patients. Follow-up data included C5 levels, intellectual quotient and correlation genotype-phenotype. IVA was detected by NBS in 8 patients (prevalence of 1/326 629). Except 1, all the 8 patients identified by NBS were asymptomatic at diagnosis and had isovalerylcarnitine (C5) levels of 1.6-6.4 µM and isovalerylglycine (IVG) levels <1100 mmol per mol creatinine; they remained asymptomatic with a natural protein intake ⩾1.5 g kg-1 per day. Symptomatic patients with chronic intermittent or acute neonatal IVA had C5 levels of 3.9-16.3 µM and IVG levels >3400 mmol per mol creatinine. The percentage of isovalerate incorporation in fibroblasts was 64-80% in patients detected by NBS and 4.9-13% in symptomatic patients. Cognitive function was within normal ranges in all patients but was negatively correlated with IVG at detection (-0.592; P<0.05). The genetic analysis revealed nine novel mutations. The clinical/biochemical phenotype correlated fairly well with the phenotype predicted by the mutations found. In conclusion, although blood C5 levels have traditionally been considered the prognostic marker of choice, urine IVG levels would appear to be a better predictor, as they correlated well with severity of mutations and were associated with a lower incorporation rate of IVA in fibroblasts and a less favorable clinical course.